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Complement C5a has been proposed as a major regulator of myeloid-derived suppressor cells (MDSCs). In this thesis, we aimed to evaluate the mechanisms by which the C5a/C5aR1 axis endows MDSCs with properties that facilitate tumor growth and metastatic spread. Local C5a production in primary tumors seems to promote the recruitment of MDSCs, which results in a decrease in the frequency and activity of CD8 T cytotoxic lymphocytes. For this reason, we hypothesized that combined inhibition of C5a/C5aR1 and programmed cell death protein 1 (PD-1) signaling may have a synergistic antitumor effect. Using syngeneic models of lung cancer, we demonstrate that the combined blockade of C5a (AON-D21) and PD-1 (RMP1-14) markedly reduced tumor growth and led to prolonged survival. This effect was accompanied by a negative association between the frequency of CD8 T cells and MDSCs within tumors, which may result in a more complete reversal of CD8 T-cell exhaustion. This study provides support for the clinical evaluation of anti PD-1 and anti-C5a drugs as a novel combination therapeutic strategy for lung cancer. We also characterized the role of C5a on the two distinct subsets of MDSCs: polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (MON-MDSCs). Using ex vivo generated MDSCs and antagonists for C5a (AON-D21) and C5aR1 (PMX53), we demonstrate that C5a promoted an integrin-independent amoeboid mode of migration on PMN-MDSCs, while had no apparent effect on MON-MDSCs. In association with the effect on PMN-MDSC migration, C5a downregulated the expression of β1 and β3 integrins (mediators of cell-matrix adhesion) and upregulated the expression of the mediator of leukocyte extravasation CD11b. Moreover, stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the extrusion of neutrophil extracellular traps (NETs). C5a/C5aR1 blockade or NET inhibition reduced the number of circulating tumor cells (CTCs) and the metastatic burden in a lung metastasis model. In support of the relevance of these findings obtained in mice, C5a was able to stimulate invasion and NETosis of PMN-MDSCs obtained from lung cancer patients. Moreover, myeloperoxidase (MPO)-DNA complexes, markers of NETosis, were elevated in lung cancer patients and correlated with C5a levels. We conclude that blockade of C5a results in a substantial improvement in the efficacy of anti PD-1 antibodies against lung cancer growth, and that C5a induces the formation of NETs by MDSCs to facilitate the dissemination, colonization and metastasis of cancer cells.

Cardiovascular diseases (CVD) are the leading cause of death in the developed countries. According to the las report of the European Society of Cardiology, CVD remain the most common cause of death within Europe, accounting for 2.2 million deaths in females and 1.9 million deaths in males. These equate to 47% and 39% of all deaths in females and males, respectively, and almost half of them are caused by Myocardial Infarction (MI). In the last 20 years, in order to overcome the limitations of the conventional treatments, cell therapy, has emerged as a promising therapeutic option for the treatment of MI. In this thesis we have employed Adipose Derived Mesenchymal Stem Cells (ADSC) seeded in a collagen scaffold fabricated under GMP-conditions, to treat the infarcted heart in preclinical MI animal models. First, we assessed the patch safety in rodent models, proving a safe profile of the system, established by tumorigenicity, toxicity and biodistribution studies. Secondly, the collagen scaffold was cellularized with allogeneic ADSC and its immunological effect was analysed both in vitro and in vivo, showing no alloreactive response by the immune cells towards the allogeneic patch. Moreover in vitro, in a human context, the cellularized patch elicited a potent immunomodulatory effect towards lymphocytes by inhibiting their proliferation, phenotypical activation and pro-inflammatory cytokine production. Finally, the functionalization of the collagen membrane with the angiogenic factor SDF-1 was achieved, by the creation of a bilayer collagen scaffold, which confirmed to be biocompatible in vivo and exerted a therapeutic effect when tested in a rat MI model in the shape of cardiac function recovery.

Polyphenols have shown ability to modulate DNA damage, inflammation, and the gut microbiota. Cardoon stalks (Cynara cardunculus var. L. altilis DC) and sweet Italian green peppers fruits (Capsicum annuum L.) are vegetables with different polyphenolic profile (chlorogenic acids vs flavonoids respectively). Thus, the main aim of this project was to evaluate the potential genoprotective and/or anti-inflammatory and prebiotic activity of cardoon stalks and sweet Italian green pepper fruits in the gastrointestinal (GI) tract, by simulating an intake situation. A total of 30 polyphenols were identified and quantified by HPLC-MS/MS in white and red cardoon stalks. Red cardoon almost doubled the polyphenol content of white cardoon, and was therefore selected for the assessment of its biological activity. Besides, the impact of 4 heat treatments (i.e., blanching, different frying conditions, traditional boiling and sous-vide cooking) on the antioxidant capacity and/or the polyphenol profile and content of cardoon stalks, was evaluated. Then, sous-vide cooking was selected for red cardoon, and griddling for green pepper, as they favour a high polyphenol bioaccessibility after GI digestion. Bioaccessible polyphenols of GI-digested green pepper did not show genoprotection against oxidatively generated damage in HT-29 cells, regardless of the application of a heat treatment (griddling). Besides, high polyphenol concentrations caused a slight pro-oxidant effect on DNA. GI-digested raw red cardoon and raw green pepper had

Myocardial infarction (MI) leads to an irreversible loss of cardiac myocytes, which compromises cardiac function. The cellular and molecular mechanisms involved in the ischemic event remain under study, being the cardiac exosomes transfer of non-coding RNAs a key process in this pathology. In this context, a comparative analysis of the exosomal compartment of human cardiac progenitor cells (CPC) was performed in comparison with human bone marrow-mesenchymal stem cells (MSC) and human dermal fibroblasts (HDF). A total of 481 differentially expressed microRNAs (miR) were found, being miR-935 the most differentially expressed in CPC. Analysis of miR-935 in vitro overexpression promoted a positive trend to increment cardiomyocyte survival, in response to oxidative stress. Furthermore, when miR-935 regulation was analyzed in a mouse model of MI disease, miR downregulation was shown five days post-infarct in different cardiac subpopulations. In order to study the role of miR-935 in MI and to achieve an optimal miR-935 overexpression in our mouse model, we developed a robust viral-based method for cardiac-specific miR overexpression. Cardiotropic Serotype 9-Adeno-Associated Virus (AAV9) were used for this purpose and their biodistribution was first determined in mice. Ubiquitous EF1α or the cardiac-specific TnT promoters were combined with Luciferase (Luc) or GFP reporters and administered by intramyocardial or intravenous injection, either in healthy or myocardial-infarcted mice. High transgene expression levels were found in the heart, but not in the liver, of mice receiving AAV-TnT, which was significantly higher after intramyocardial injection regardless of ischemia-induction. On the contrary, high hepatic transgene expression levels were detected with the EF1α-promoter, independently of the administration route and heart damage. Luc expression increased with both promoters in a time-dependent manner, reaching a peak by day 3-7 that was stable for at least 60 days. Moreover, tissue-specific GFP expression was found in cardiomyocytes with the TnT-vector, while minimal cardiac expression was detected with the ubiquitous one. Interestingly, we found that MI greatly increased the transcriptional activity of AAV genomes. Thus, we found AAV9-TnT as a robust and stable vector for cardiac-specific delivery after intramyocardial injection. Finally, the therapeutic potential of this vector in combination with miR-935 was evaluated in a mouse model of myocardial ischemia. Intramyocardial injection of AAV9-TnT-miR935 vector did not significantly improve heart function 60 days post-infarct. However, a slight positive trend in the ejection fraction and a decreased adverse cardiac remodeling are observed, suggesting miR-935 putative cardioprotective role.

Stem cells allow to investigate about the basic mechanisms that regulate embryonic development, cellular plasticity, and organ maintenance and regeneration. Induced pluripotent stem cells (iPSCs) are a powerful source of cells for diverse applications such as developmental and disease modeling, drug discovery and regenerative medicine. Cardiac development is coordinated by complex interactions between cardiac progenitor cell populations, different molecular signaling pathways, and spatially and temporally regulated gene expression. Cardiovascular progenitors (CVPs), with similar potential to these present in early stages of embryonic development, can be obtained from iPSCs by mimicking signaling during cardiogenesis, creating an ideal cell source to treat the damaged heart. However, the mechanisms and conditions for long-term self-renewal and maintenance of CVPs remain elusive. The generation of new iPSC models for tracing CVP lineages permits to delve into the biology of CVPs and discover novel potential regulators of their fate. We have established three different Cre/LoxP mouse models for lineage tracing of CVPs and their cell progeny by the expression of ZsGreen (ZsG) protein: Ai6-Mesp1-Cre (Mesp1 tracer), Ai6-Isl1-Cre (Isl1 tracer) and Ai6-Mef2c-AHF-Cre (AHF tracer) mice. Multiple iPSC clones have been derived from Ai6-Isl1-Cre and Ai6-Mef2c-AHF-Cre reporter mice. Several generated iPSC lines have been fully characterized, demonstrating embryonic stem-like features. iPSCs encoded the expected genomic insertions, showed normal karyotypes, transgenes were silenced, and expressed endogenous pluripotency-associated markers. Moreover, iPSCs were capable to differentiate into the three germ layers both in vitro and in vivo. We have verified the utility of established AHFiPSCs to track CVPs and their differentiated progeny. Upon differentiation, ZsG+ cells derived from AHFiPSCs appeared from embryoid body (EB) day 6 onwards, expressed cardiovascular-related markers, and were able to differentiate into cardiomyocytes, endothelial and smooth muscle cells. Comparative gene expression analysis using four different AHFiPSC lines revealed distinct molecular signatures in three particular stages of differentiation: undifferentiated iPSCs (AHFiPS-D0), sorted ZsG+ cells at day 6 (AHFiPS-D6.ZsG+) and sorted ZsG+ cells at day 13 of differentiation (AHFiPS-D13.ZsG+), that expressed pluripotency-, CVP- and cardiac/vascular lineages-associated markers, respectively. Gapdh and Polr2a have been determined the most stable housekeeping genes along the differentiation of AHFiPSCs, being optimal for accurate normalization of gene expression in our samples. We have identified novel regulators of transcription specifically upregulated in CVPs: Lin28a (and its paralog Lin28b), Lhx1 and Nr6a1. The expression of these genes was also found increased in the corresponding CVP-enriched samples from two different public analyses using mouse and human pluripotent stem cells. Moreover, using bioinformatic analysis of biological pathways we have found p53 as an interconnecting molecule of all these selected regulators of transcription. In order to explore novel insights into the biological role of the selected regulators in CVP fate, we have generated an inducible vector (pTRE-CDS-IRES-Puro-REX1-Blast) to carry out gain-of-function (GOF) analyses. This Tet-On system worked properly in AHFiPSC clones, but unfortunately it failed to work in EBs of certain size along differentiation. In contrast, this GOF system correctly functioned in human iPSCs differentiated in monolayer cultures. We have established four CBiPS1sv-4F-5 cell lines carrying Tet-On systems for the inducible expression of LIN28A, LIN28B, NR6A1 and LHX1, and preliminary results indicated that these regulators of transcription might have a role in CVP fate determination.

Los objetivos de esta tesis doctoral son: 1) Evaluar el papel del FGF15/19 endógeno en respuesta a la administración de una dieta rica en grasa. 2) Desarrollar una nueva proteína basada en la fusión del FGF19 y la ApoA-I que aumente la vida media y el tropismo hepático. 3) Evaluar la actividad biológica de la nueva proteína de fusión, así como su posible aplicación en modelos experimentales en los que la regeneración hepática esté impedida.

The tumor microenvironment is characterized to be under severe conditions (nutrient shortage, low pH and oxygen deprivation) that affect not only tumor cells but also stroma and immune infiltrate. Tumor cells have developed mechanism in order to adapt their biology to such extreme circumstances. One of these adaptation strategies is the HIF-1α pathway that is activated under hypoxia (low oxygen availability) promoting angiogenesis, metabolic changes and survival. The projects that have been carried out during this thesis are focused on the influence of hypoxia in the biology of malignant and immune cells in the tumor microenvironment regarding the regulation of two important lymphocyte receptors: CD137 and CD69. Under hypoxic conditions tumor cells promote the alternative splicing of the co-stimulatory receptor CD137 (also known as 4-1BB) resulting in the generation of a functional soluble form of CD137 (sCD137). The secreted sCD137 binds the natural ligand of CD137 (present on APCs) preventing the CD137 ligand-mediated co-stimulation of T-lymphocytes acting as an immune-evasion mechanism. Additionally, CD137 silencing in a colon carcinoma cell line enhances the tumor growth control mediated by CD8+ T lymphocytes. Tumor-infiltrating T lymphocytes (TILs) are also influenced by hypoxia that controls many of the mechanisms that confer T-cells a cytotoxic phenotype. TILs from transplantable and spontaneous mouse tumors express the early T-cell activation marker CD69 more intensely than lymphocytes located in a non-hypoxic tissue such as the spleen. The second study of this thesis points out that hypoxia up-regulates CD69 in T-lymphocytes undergoing TCR-CD3 activation in a HIF-1α dependent manner. Indeed, HIF-1α is able to bind a hypoxia response element (HRE) that we have found in the promoter of the human CD69 gene. In addition, in vivo staining with pimonidazole shows a correlation between hypoxia and the intensity of CD69 expression on T lymphocytes from hypoxic tissues, such as bone marrow and tumor. These studies highlight that hypoxia plays an important role in the interplay between tumors and immune infiltrates in the tumor microenvironment.